To fully express its pathogenic potential, HIV-1 must successfully replicate in two different cellular environments, namely proliferating CD4+ T cells and non-dividing CD4+ macrophages. Most retroviruses fail to grow in postmitotic cells because they are unable to gain entry into the nucleus through the nuclear pore. However, HIV-1 encodes three karyophilic proteins: Vpr, Integrase, and Matrix, which effectively promote nuclear uptake of the viral preintegration complex permitting this virus to grow in such non-dividing cells. -The HIV preintegration complex corresponds to one of the largest inbound cargoes successfully handled by the nuclear pore complex. Vpr plays a particularly important role in the replication of HIV in macrophages, likely reflecting its karyophilic properties. Our studies now indicate that Vpr contains at least two noncanonical nuclear localization signals that access two novel pathways of nuclear import. Specific Aim l of this proposal seeks to understand in more precise terms the biochemical basis for Vpr-mediated nuclear uptake including the nature of the targeting signals, the receptors recognized by each, and the role of each of these signals in nuclear import of the HIV preintegration complex in primary human macrophages. In addition to its karyophilic properties, HIV-1 Vpr also blocks the proliferation of human CD4+ T cells by arresting or delaying their passage through the G2 checkpoint of the cell cycle. Thus, unlike other retroviruses, HIV-1 induces an activated but nonproliferating cellular environment for its optimal replication. Specific Aim 2 of this proposal seeks to delineate the molecular mechanism(s) underlying the G2-arresting properties of Vpr, including identification of the key host cofactors targeted by Vpr. These studies will also explore whether G2 arrest occurs in HIV-infected patients. By more completely understanding how Vpr functions in the HIV infected host cell, we hope to better understand the nature of the interplay of this virus with its host, to gain new insights into the biology of normal nuclear import and cell cycle control, and possibly to develop new approaches to interfering with the pathogenic effects of HIV infection.